PROJECT SUMMARY The frequency of infection and the number of deaths due to malaria are staggering with over 200 million cases each year, and nearly one million deaths, mostly among children. While the symptoms of malaria can be quite severe, asymptomatic malaria cases account for ~50-90% of infections. Asymptomatic malaria can last for months, and contributes to propagation of disease as these individuals remain infectious to the mosquito vector. These cases also complicate eradication efforts as the number of parasites circulating in the blood frequently falls below the limit of detection of blood smears, the gold standard for malaria diagnostics. Here, our goal is to develop a method to cryogenically enrich parasites from blood to enhance the sensitivity of microscopy and enable identification of parasites among asymptomatic individuals. One confounding factor in cryobiology is that each unique cell line requires its own unique cryopreservation protocol. This is particularly troublesome for the cryopreservation of non-homogenous cells and tissues. Here, our goal is to exploit this principle of low temperature biology in order to selectively cryopreserve malaria parasites while destroying uninfected blood cells, thereby achieving a `cryogenic enrichment' to enhance microscopic detection of low-parasitemia infections. Throughout this proposal, emphasis is given to methods that are low-cost, simple and easy to implement into the existing infrastructure to ensure smooth translation to point-of-care. In Specific Aim 1, we will optimize the cryogenic enrichment protocol to improve the morphology, recovery and purity of malaria parasites. This will be achieved by quantifying the transport properties of a range of cryoprotectants in purified blood cells (i.e. uninfected erythrocytes, leukocytes and malaria parasites) to identify conditions where optimal permeability (and hence cell protection during cryopreservation) occurs in parasites, but not for other blood cells. In Specific Aim 2, we will determine the feasibility of cryogenic enrichment for field implementation based upon i) cooling requirements for freezing cells, ii) scalability and sensitivity and iii) compatibility with alternative malaria diagnostic methods. Our ultimate goal is to develop a simplified and sustainable cryogenic enrichment protocol to enhance the sensitivity of microscopy for malaria diagnosis in a manner that is readily accessible to labs and clinics worldwide.